Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is designed for the in vitro modeling of human cervicovaginal mucus. Vag3Gel is presented here as a platform for culturing human microbiota samples and single strains. The protocol describes how to culture cervicovaginal microbiota and single strain samples in Vag3Gel using a syringe format and how to prepare the samples for downstream analyses. Downstream assays include cell viability assessment, flow cytometry, metabolomics, and sequencing. The potential applications of Vag3Gel include microbial mining, drug screening, and permeability assays.
3x syringes, each containing 5 mL Vag3Gel (Ovulatory/Non-ovulatory/Pregnancy).
50 mL Bac3Gel® dissolution medium (200 mM sodium citrate).
Material/Reagent Required But Not Provided
Syringe connector.
0.9% (w/v) NaCl solution prepared in deionized water.
MRS (de Man, Rogosa, and Sharpe) medium.
ImportantImportant
MRS medium is only required if the acquired Vag3Gel was not produced in MRS.
Storage Conditions
Store in a dry place inside tightly sealed containers at 2-8 °C.
This product can be stored for at least 9 months.
Precautions and Disclaimer
For R&D use only.
Procedure
Before you begin
The protocols below describe the specific steps for culturing cervicovaginal microbiota samples and single strain samples. However, these protocols have also been used to culture co-cultures, and microbial consortia. The Vag3Gel used in these protocols is composed of 2.5, 3.0, or 5.0 mg.mL-1 porcine stomach type III mucin for Ovulatory, Non-ovulatory and Pregnancy versions, respectively, and 0.07 mg.mL-1 NaCl or MRS medium. Vag3Gel’s composition is fully tunable; customized compositions are commercially available (i.e. incorporation of other mucin types, proteins, lipids and media).
Ensure all instruments and consumables (menstrual cup, pipettes, falcons, syringes, syringe connector, and microcentrifuge tubes) are sterilized and free of contaminants.
All manipulations should be performed under aseptic conditions, ideally in a Class II biological safety cabinet.
Prepare a 0.9% (w/v) sterile NaCl solution.
Vag3Gel largely favors facultative anaerobes when incubated under aerobic conditions. Its use is especially recommended for culturing Lactobacillaceae.
Select a protocol:
A. Cervicovaginal Sample Isolation and Processing
Timing: ~ 75 min
1. The donor should use a menstrual cup for 60 minutes.
NoteNote
The menstrual cup must be handled exclusively with gloves. Gloves should be cleaned with a 70% ethanol wipe before handling the menstrual cup.
After use, recover the menstrual cup and transfer it into a sterile 50 mL Falcon tube.
2. Centrifuge the Falcon tube containing the menstrual cup and its contents at 8,000 xg for 15 minutes to collect the secretions.
Discard the menstrual cup after centrifugation.
Resuspend the recovered sample in up to 100 mL of sterile MRS medium within a sterile Schott flask.
ImportantImportant
If Vag3Gel was produced in MRS, researchers should resuspend the recovered sample in 0.9 (w/v) NaCl instead of MRS medium.
Homogenize the suspension by gently inverting the flask several times and pipetting up and down.
WarningCRITICAL
Cervicovaginal samples should be as fresh as possible; ideally, processing should start within 30 minutes from collection time and kept on ice.
Unused cervicovaginal samples can be aliquoted, supplemented with glycerol (final concentration of 10% v/v), and stored at -80 °C for future experiments. This should be done as quickly as possible to preserve microbial diversity.
B. Culturing in Vag3Gel
Timing: ~ 5 minutes
1. Remove the syringe cap and keep it sterile.
2. Discard the first 2-3 drops of Vag3Gel by gently pressing the syringe plunger.
3. Using the double syringe mixing method, combine the inoculum and Vag3Gel in a 1:1 volume ratio by mixing 6-7 times.
4. Insert the cap back into the syringe containing the inoculum + Vag3Gel.
5. Incubate the syringe at 37 °C for up to 72 hours under aerobic conditions, according to the experimental design.
NoteNote
The recommended incubation period is 72 hours but it may be adjusted as required by the user.
To test the effects of molecules (e.g. biotics, active principles) on microbiota, it is recommended to let microbiota stabilize in Vag3Gel for 24 hours at 37 °C before adding the molecule of interest using the double syringe mixing method.
WarningCRITICAL
While not a strict requirement, ensuring anaerobic conditions prior culturing in Vag3Gel will prevent the loss of obligate anaerobes.
C. Dissolution of Vag3Gel
Timing: ~ 5 min per syringe
1. Add Bac3Gel® dissolution medium (200 mM sodium citrate solution) in a 1:1 ratio to each sample. Mix thoroughly to fully dissolve Vag3Gel structure.
The homogenized samples can be used for downstream assays.
NoteNote
For sequencing: Centrifuge the samples (14,000 x g, 5 minutes), then use the pellet for DNA extraction.
For metabolomics: Centrifuge the samples (14,000 x g, 10 minutes), then collect and lyophilize the supernatant.
For measuring metabolic activity or optical density: Centrifuge the samples (14,000 xg, 5 minutes), discard the supernatant, then resuspend in 0.9 (w/v) NaCl.
Other applications include assessing cell viability, performing flow cytometry analysis, and conducting standard microbiological characterization assays.
A. Preparation of Microbial Suspensions
Timing: ~ 15 min
1. Grow cultures of the species of interest (e.g. Lactobacillus) to the logarithmic (log) growth phase in the appropriate medium.
Centrifuge the cultures to pellet the cells, then discard the supernatant.
Resuspend the cell pellet in an appropriate culture medium.
ImportantImportant
If Vag3Gel was produced in MRS, researchers should resuspend the cell pellet in 0.9 (w/v) NaCl instead of MRS medium.
Measure the optical density at 600 nm (OD600) of the microbial suspension.
Adjust the cell concentration as needed.
B. Culturing in Vag3Gel
Timing: 5 minutes
1. Discard the first 2-3 drops of Vag3Gel by gently pressing the syringe plunger.
2. Using the double syringe mixing method, combine the inoculum and Vag3Gel in a 1:1 volume ratio by mixing 6-7 times.
Dispense the syringe contents into a 15 mL falcon tube. Using a positive displacement pipette distribute the sample in equal volumes into microcentrifuge tubes.
NoteNote
If a syringe connector is not available, follow steps 3 and 4 instead, otherwise skip to step 5 after completing this step.
3. Dispense Vag3Gel from the syringe into a sterile 15 mL falcon tube.
4. Using a positive displacement pipette, transfer 500 μL of Vag3Gel into microcentrifuge tubes.
NoteNote
Instead of microcentrifuge tubes, researchers can use 24-well plates.
Add an equal volume (500 μL) of the microbial suspensions prepared in A. and homogenize the mixture by gently pipetting.
5. Incubate the microcentrifuge tubes/24-well plate at 37 °C for up to 72 hours under aerobic or anaerobic conditions, according to the experimental design.
